The focus of the current proposal is the Na/dicarboxylate cotransporters of the renal proximal tubule, particularly NaDC-1, the low affinity transporter of the apical membrane. NaDC-1 plays an important role in the absorption of Krebs cycle intermediates, such as citrate and succinate. The transport activity of NaDC-1 affects physiological functions such as citrate homeostasis in the urine (which influences the development of kidney stones), organic anion secretion, maintenance of acid-base balance, and overall metabolic status of the body. The long-term objective of this research is to determine the structural basis of ion and substrate binding and translocation in the Na+/dicarboxylate cotransporters to better understand the mechanism of ion-coupled transport. The results generated in the current grant period have identified several key amino acids that may participate in substrate and cation binding and may form part of the permeation pathway through the protein. In the first specific aim of this proposal, we plan to identify additional residues and domains in the NaDC-1 transporters that determine specific functional properties, such as substrate and cation selectivity and affinity, and sensitivity to inhibitors. In the second specific aim, we plan to use the substituted cysteine-accessibility method to identify the ion and substrate permeation pathways in NaDC-1. In the third aim, we plan to refine the secondary structure model of NaDC-1. The studies outlined in this proposal should provide fundamental information on the functional properties of the NaDC-related family of sodium-coupled transporters and on the physiological role of these transporters in the kidney.